One assay to test them all: Multiplex assays for expansion of respiratory virus surveillance.

Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Clinical performance of XpertXpress® SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath™ COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek™ SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1 to 2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97 and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhance diagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections.

Development, Validation, and Utilization of a Luminex-Based SARS-CoV-2 Multiplex Serology Assay.

SARS-CoV-2 antibody testing is important for seroprevalence studies and for evaluating vaccine immune responses. We developed and validated a Luminex bead-based multiplex serology assay for measuring IgG levels of anti-SARS-CoV-2 antibodies against full-length spike (S), nucleocapsid (N), and receptor-binding domains (RBDs) of wild-type, RBD N501Y mutant, RBD E484K mutant, RBD triple mutant SARS-CoV-2 proteins, Sars-CoV-1, MERS-CoV, and common human coronaviruses, including SARS-CoV-2, OC43, 229E, HKU1, and NL63. Assay cutoff values, sensitivity, and specificity were determined using samples from 160 negative controls and 60 PCR-confirmed, SARS-CoV-2-infected individuals. The assay demonstrated sensitivities of 98.3%, 95%, and 100% and specificities of 100%, 99.4%, and 98.8% for anti-(S), -N, and -RBD, respectively. Results are expressed as IgG antibody concentrations in BAU/mL, using the WHO international standard (NIBSC code 20/136) for anti-SARS-CoV-2 IgG antibodies. When the multiplex assay was performed and compared with singleplex assays, the IgG antibody measurement geometric mean ratios were between 0.895 and 1.122, and no evidence of interference was observed between antigens. Lower and upper IgG concentration limits, based on accuracy (between 80% and 120%), precision (percent relative standard deviation, ≤25%), and sample dilutional linearity (between 75% and 125%), were used to establish the assay range. Precision was established by evaluating 24 individual human serum samples obtained from vaccinated and SARS-CoV-2-infected individuals. The assay provided reproducible, consistent results with typical coefficients of variation of ≤20% for all assays, irrespective of the run, day, or analyst. Results indicate the assay has high sensitivity and specificity and thus is appropriate for use in measuring SARS-CoV-2 IgG antibodies in infected and vaccinated individuals. IMPORTANCE The SARS-CoV-2 pandemic resulted in the development and validation of multiple serology tests with variable performance. While there are multiple SARS-CoV-2 serology tests to detect SARS-CoV-2 antibodies, the focus is usually either on only one antigen at a time or multiple proteins from only one SARS-CoV-2 variant. These tests usually do not evaluate antibodies against viral proteins from different SARS-CoV-2 variants or from other coronaviruses. Here, we evaluated a multiplex serology test based on Luminex technology, where antibodies against multiple domains of SARS-CoV-2 wild type, SARS-CoV-2 mutants, and common coronavirus antibodies are detected simultaneously in a single assay. This Luminex-based multiplex serology assay can enhance our understanding of the immune response to SARS-CoV-2 infection and vaccination.

Methodological approaches to optimize multiplex oral fluid SARS-CoV-2 IgG assay performance and correlation with serologic and neutralizing antibody responses.

BACKGROUND: Oral fluid (hereafter, saliva) is a non-invasive and attractive alternative to blood for SARS-CoV-2 IgG testing; however, the heterogeneity of saliva as a matrix poses challenges for immunoassay performance. OBJECTIVES: To optimize performance of a magnetic microparticle-based multiplex immunoassay (MIA) for SARS-CoV-2 IgG measurement in saliva, with consideration of: i) threshold setting and validation across different MIA bead batches; ii) sample qualification based on salivary total IgG concentration; iii) calibration to U.S. SARS-CoV-2 serological standard binding antibody units (BAU); and iv) correlations with blood-based SARS-CoV-2 serological and neutralizing antibody (nAb) assays. METHODS: The salivary SARS-CoV-2 IgG MIA included 2 nucleocapsid (N), 3 receptor-binding domain (RBD), and 2 spike protein (S) antigens. Gingival crevicular fluid (GCF) swab saliva samples were collected before December 2019 (n = 555) and after molecular test-confirmed SARS-CoV-2 infection from 113 individuals (providing up to 5 repeated-measures; n = 398) and used to optimize and validate MIA performance (total n = 953). Combinations of IgG responses to N, RBD and S and total salivary IgG concentration (µg/mL) as a qualifier of nonreactive samples were optimized and validated, calibrated to the U.S. SARS-CoV-2 serological standard, and correlated with blood-based SARS-CoV-2 IgG ELISA and nAb assays. RESULTS: The sum of signal to cutoff (S/Co) to all seven MIA SARS-CoV-2 antigens and disqualification of nonreactive saliva samples with ≤15 µg/mL total IgG led to correct classification of 62/62 positives (sensitivity [Se] = 100.0%; 95% confidence interval [CI] = 94.8%, 100.0%) and 108/109 negatives (specificity [Sp] = 99.1%; 95% CI = 97.3%, 100.0%) at 8-million beads coupling scale and 80/81 positives (Se = 98.8%; 95% CI = 93.3%, 100.0%] and 127/127 negatives (Sp = 100%; 95% CI = 97.1%, 100.0%) at 20-million beads coupling scale. Salivary SARS-CoV-2 IgG crossed the MIA cutoff of 0.1 BAU/mL on average 9 days post-COVID-19 symptom onset and peaked around day 30. Among n = 30 matched saliva and plasma samples, salivary SARS-CoV-2 MIA IgG levels correlated with corresponding-antigen plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.83, S: ρ = 0.82; all p < 0.001). Correlations of plasma SARS-CoV-2 nAb assay area under the curve (AUC) with salivary MIA IgG (N: ρ = 0.68, RBD: ρ = 0.78, S: ρ = 0.79; all p < 0.001) and with plasma ELISA IgG (N: ρ = 0.76, RBD: ρ = 0.79, S: ρ = 0.76; p < 0.001) were similar. CONCLUSIONS: A salivary SARS-CoV-2 IgG MIA produced consistently high Se (> 98.8%) and Sp (> 99.1%) across two bead coupling scales and correlations with nAb responses that were similar to blood-based SARS-CoV-2 IgG ELISA data. This non-invasive salivary SARS-CoV-2 IgG MIA could increase engagement of vulnerable populations and improve broad understanding of humoral immunity (kinetics and gaps) within the evolving context of booster vaccination, viral variants and waning immunity.

A multiplex assay for the detection of antibodies to relevant swine pathogens in serum.

Livestock industry supports the livelihood of around 1.3 billion people in the world, with swine industry contributing with 30% of total livestock production worldwide. To maintain and guarantee this production, a pivotal point according to the OIE is addressing potential biohazards. To control them, permanent sero-surveillance is crucial to achieve more focused veterinary public health intervention and prevention strategies, to break the chains of transmission, and to enable fast responses against outbreaks. Within this context, multiplex assays are powerful tools with the potential to simplify surveillance programs, since they reduce time, labour, and variability within analysis. In the present work, we developed a multiplex bead-based assay for the detection of specific antibodies to six relevant pathogens affecting swine: ASFV, CSFV, PRRSV, SIV, TB and HEV. The most immunogenic target antigen of each pathogen was selected as the target protein to coat different microsphere regions in order to develop this multiplex assay. A total of 1544 serum samples from experimental infections as well as field samples were included in the analysis. The 6-plex assay exhibited credible diagnostic parameters with sensitivities ranging from 87.0% to 97.5% and specificities ranging from 87.9% to 100.0%, demonstrating it to be a potential high throughput tool for surveillance of infectious diseases in swine.

Challenges of Multiplex Assays for COVID-19 Research: A Machine Learning Perspective.

Multiplex assays that provide simultaneous measurement of multiple analytes in biological samples have now developed into widely used technologies in the study of diseases, drug discovery, and other medical areas. These approaches span multiple assay systems and can provide readouts of specific assay components with similar accuracy as the respective single assay measurements. Multiplexing allows the consumption of lower sample volumes, lower costs, and higher throughput compared with carrying out single assays. A number of recent studies have demonstrated the impact of multiplex assays in the study of the SARS-CoV-2 virus, the infectious agent responsible for the current COVID-19 pandemic. In this respect, machine learning techniques have proven to be highly valuable in capturing complex disease phenotypes and converting these insights into models which can be applied in real-world settings. This chapter gives an overview of opportunities and challenges of multiplexed biomarker analysis, with a focus on the use of machine learning aimed at identification of biological signatures for increasing our understanding of COVID-19 disease, and for improved diagnostics and prediction of disease outcomes.

N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes.

Studies conducted using murine arthritis models have indicated that the use of in vitro-transcribed messenger RNA (IVT mRNA) is an effective therapeutic approach for joint diseases. However, the use of IVT mRNA in human synovial cells has not been widely studied. Recently, the outbreak of the novel coronavirus disease has accelerated the development of innovative mRNA vaccines, such as those containing a modified nucleic acid, N1-methylpseudouridine-5'-triphosphate (m1ψ). IVT mRNA is an attractive tool for biological experiments and drug discovery. To verify the protein expression from IVT mRNA in vitro, primary cultured fibroblast-like synoviocytes (FLS) and MH7A human synovial fibroblast cells were transfected with enhanced green fluorescent protein (EGFP) mRNA with or without m1ψ incorporation. EGFP was detected using western blotting and fluorescence microscopy. A multiplex assay was performed to comprehensively understand IVT mRNA-induced immunogenicity. Gene expression levels were measured using reverse transcription polymerase chain reaction. In both MH7A cells and FLS, cells transfected with EGFP mRNA containing m1ψ generated higher levels of EGFP than those transfected with unmodified EGFP or control mRNAs. The multiplex assay of the FLS culture supernatant and reverse transcription polymerase chain reaction for FLS revealed that both concentration and expression of IL-6, TNF-α, and CXCL10 were upregulated by unmodified EGFP mRNA, whereas they were suppressed by EGFP mRNA with m1ψ. Overall, m1ψ incorporation enhanced protein expression and decreased the expression of cytokines. These findings may contribute to arthritis research.

SARS-CoV-2 RNA Detection on Environmental Surfaces in a University Setting of Central Italy.

The transmission of SARS-CoV-2 occurs through direct contact (person to person) and indirect contact by means of objects and surfaces contaminated by secretions from individuals with COVID-19 or asymptomatic carriers. In this study, we evaluated the presence of SARS-CoV-2 RNA on surfaces made of different materials located in university environments frequented by students and staff involved in academy activity during the fourth pandemic wave (December 2021). A total of 189 environmental samples were collected from classrooms, the library, computer room, gym and common areas and subjected to real-time PCR assay to evaluate the presence of SARS-CoV-2 RNA by amplification of the RNA-dependent RNA polymerase (RdRp) gene. All samples gave a valid result for Internal Process Control and nine (4.8%) tested very low positive for SARS-CoV-2 RNA amplification with a median Ct value of 39.44 [IQR: 37.31-42.66] (≤1 copy of viral genome). Our results show that, despite the prevention measures implemented, the presence of infected subjects cannot be excluded, as evidenced by the recovery of SARS-CoV-2 RNA from surfaces. The monitoring of environmental SARS-CoV-2 RNA could support public health prevention strategies in the academic and school world.

Exploratory assessment of serological tests to determine antibody titer against SARS-CoV-2: Appropriateness and limits.

BACKGROUND: Serological tests can be used to detect antibodies in the serum of subject's after SARS-CoV-2 infection and vaccination. Currently, variability in antibody titers and the availability of a multiplicity of serological tests have made it necessary to highlight their appropriateness and limitations in various diagnostic settings. METHODS: This study is part of Covidiagnostix, a multicenter project aimed at the assessment of the health technology used in SARS-CoV-2 serological tests. Based on data gained from the analysis of over 5000 subjects, a selected number of serum samples, representative of different diagnostic settings, were analyzed first by qualitative immunoassays (IgA, M, and G MILLIPLEX® SARS-CoV-2 tests based on Luminex® ) to define the immunoglobulins serum composition and subsequently by four serological diagnostic tests (Elecsys Anti-SARS-CoV-2 and Elecsys Anti-SARS-CoV-2 S by Roche, SARS-CoV-2 IgG by Siemens Healthcare, and CHORUS SARS-CoV-2 "NEUTRALIZING" Ab by DIESSE). The first WHO International Standard for SARS-CoV-2 was also analyzed using the same methods. RESULTS: This study evaluated the antibody content and titer of the WHO Standard and serum of subjects with/without previous infection and before/after vaccination for SARS-CoV-2. CONCLUSION: The definition of antibodies in the WHO standard and the analysis of serum samples allowed for the identification of the appropriateness of serological tests in each diagnostic setting, increasing the effectiveness of the resulting laboratory data. Furthermore, we found that it would be optimal to produce new international standards against the S1 domain and RBD of the SARS-CoV-2 spike protein for a more effective serological monitoring of vaccination.

Role of immune mediators in predicting hospitalization of SARS-CoV-2 positive patients.

BACKGROUND: Several immune mediators (IM) including cytokines, chemokines, and their receptors have been suggested to play a role in COVID-19 pathophysiology and severity. AIM: To determine if early IM profiles are predictive of clinical outcome and which of the IMs tested possess the most clinical utility. METHODS: A custom bead-based multiplex assay was used to measure IM concentrations in a cohort of SARS-CoV-2 PCR positive patients (n = 326) with varying disease severities as determined by hospitalization status, length of hospital stay, and survival. Patient groups were compared, and clinical utility was assessed. Correlation plots were constructed to determine if significant relationships exist between the IMs in the setting of COVID-19. RESULTS: In PCR positive SARS-CoV-2 patients, IL-6 was the best predictor of the need for hospitalization and length of stay. Additionally, MCP-1 and sIL-2Rα were moderate predictors of the need for hospitalization. Hospitalized PCR positive SARS-CoV-2 patients displayed a notable correlation between sIL-2Rα and IL-18 (Spearman's ρ = 0.48, P=<0.0001). CONCLUSIONS: IM profiles between non-hospitalized and hospitalized patients were distinct. IL-6 was the best predictor of COVID-19 severity among all the IMs tested.

Highly Selective Multiplex Quantitative Polymerase Chain Reaction with a Nanomaterial Composite Hydrogel for Precise Diagnosis of Viral Infection.

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.

SARS-CoV-2 detection in wastewater using multiplex quantitative PCR.

A multiplex reverse transcription quantitative PCR (RT-qPCR)-based method was designed for the simultaneous detection of different SARS-CoV-2 genes. In this study, we used three target genes encoding for the nucleocapsid 1 and 3 (N1, N3), and the spike (S) proteins, all commonly used in the detection of SARS-CoV-2 in human and environmental samples. The performance of the multiplex assay, compared to the single assay was assessed for the standard calibration curve, required for absolute quantification, and then, for the real environmental samples to detect SARS-CoV-2. For this latter, four environmental samples were collected at a local wastewater treatment plant (WWTP). The results showed that the cycle threshold (Ct) values of the multiplex were comparable to the values obtained by the singleplex PCR. The amplification of the three target genes indicated the presence of SARS-CoV-2 in the four water samples with an increasing trend in February and these results were confirmed in the multiplex approach, showing the robustness of this method and its applicability for the relative abundance analysis among the samples. Overall, both the laboratory and field work results demonstrated that the multiplex PCR assay developed in this study could provide a method for SARS-CoV-2 detection as robust as the single qPCR, but faster and cost-effective, reducing by three times the number of reactions, and consequently the handling time and reagents.

A single snapshot multiplex immunoassay platform utilizing dense test lines based on engineered beads.

Co-circulation of coronavirus disease 2019 (COVID-19) and dengue fever has been reported. Accurate and timely multiplex diagnosis is required to prevent future pandemics. Here, we developed an innovative microfluidic chip that enables a snapshot multiplex immunoassay for timely on-site response and offers unprecedented multiplexing capability with an operating procedure similar to that of lateral flow assays. An open microchannel assembly of individually engineered microbeads was developed to construct nine high-density test lines, which can be imaged in a 1 mm2 field-of-view. Thus, simultaneous detection of multiple antibodies would be achievable in a single high-resolution snapshot. Next, we developed a novel pixel intensity-based imaging process to distinguish effective and non-specific fluorescence signals, thereby improving the reliability of this fluorescence-based immunoassay. Finally, the chip specifically identified and classified random combinations of arbovirus (Zika, dengue, and chikungunya viruses) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies within 30 min. Therefore, we believe that this snapshot multiplex immunoassay chip is a powerful diagnostic tool to control current and future pandemics.

IgG and IgM antibody formation to spike and nucleocapsid proteins in COVID-19 characterized by multiplex immunoblot assays.

BACKGROUND: Rapid and simple serological assays for characterizing antibody responses are important in the current COVID-19 pandemic caused by SARS-CoV-2. Multiplex immunoblot (IB) assays termed COVID-19 IB assays were developed for detecting IgG and IgM antibodies to SARS-CoV-2 virus proteins in COVID-19 patients. METHODS: Recombinant nucleocapsid protein and the S1, S2 and receptor binding domain (RBD) of the spike protein of SARS-CoV-2 were used as target antigens in the COVID-19 IBs. Specificity of the IB assay was established with 231 sera from persons with allergy, unrelated viral infections, autoimmune conditions and suspected tick-borne diseases, and 32 goat antisera to human influenza proteins. IgG and IgM COVID-19 IBs assays were performed on 84 sera obtained at different times after a positive RT-qPCR test from 37 COVID-19 patients with mild symptoms. RESULTS: Criteria for determining overall IgG and IgM antibody positivity using the four SARS-CoV-2 proteins were developed by optimizing specificity and sensitivity in the COVID-19 IgG and IgM IB assays. The estimated sensitivities and specificities of the COVID-19 IgG and IgM IBs for IgG and IgM antibodies individually or for either IgG or IgM antibodies meet the US recommendations for laboratory serological diagnostic tests. The proportion of IgM-positive sera from the COVID-19 patients following an RT-qPCR positive test was maximal at 83% before 10 days and decreased to 0% after 100 days, while the proportions of IgG-positive sera tended to plateau between days 11 and 65 at 78-100% and fall to 44% after 100 days. Detection of either IgG or IgM antibodies was better than IgG or IgM alone for assessing seroconversion in COVID-19. Both IgG and IgM antibodies detected RBD less frequently than S1, S2 and N proteins. CONCLUSIONS: The multiplex COVID-19 IB assays offer many advantages for simultaneously evaluating antibody responses to different SARS-CoV-2 proteins in COVID-19 patients.

Multiplex bead binding assays using off-the-shelf components and common flow cytometers.

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.

Multiplex Screening Assay for Identifying Cytotoxic CD8+ T Cell Epitopes.

The cytotoxicity of epitope-specific CD8+ T cells is usually measured indirectly through IFNγ production. Existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. Here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of CD8+ T cells in a single reaction. This can greatly reduce the amount of starting clinical materials for a systematic screening of CD8+ T cell epitopes. In addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of CD8+ T cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. This technique is shown to be useful to study both human and mouse CD8+ T cells. Besides, our results from human PBMCs and three independent infectious animal models (MERS, influenza and malaria) further reveal that IFNγ expression by epitope-specific CD8+ T cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). Overall, our approach opens up new possibilities for comprehensive analyses of CD8+ T cell cytotoxicity in a practical manner.

Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity.

Measurements of multiple biomolecules within the same biological sample are important for many clinical applications to enable accurate disease diagnosis or classification. These disease-related biomarkers often exist at very low levels in biological fluids, necessitating ultrasensitive measurement methods. Single-molecule arrays (Simoa), a bead-based digital enzyme-linked immunosorbent assay, is the current state of the art for ultrasensitive protein detection and can detect sub-femtomolar protein concentrations, but its ability to achieve high-order multiplexing without cross-reactivity remains a challenge. Here, a sequential protein capture approach for multiplex Simoa assays is implemented to eliminate cross-reactivity between binding reagents by sequentially capturing each protein analyte and then incubating each capture bead with only its corresponding detection antibody. This strategy not only reduces cross-reactivity to background levels and significantly improves measurement accuracies, but also enables higher-order multiplexing. As a proof of concept, the sequential multiplex Simoa assay is used to measure five different cytokines in plasma samples from Coronavirus Disease 2019 (COVID-19) patients. The ultrasensitive sequential multiplex Simoa assays will enable the simultaneous measurements of multiple low-abundance analytes in a time- and cost-effective manner and will prove especially critical in many cases where sample volumes are limited.